Lactoferrin as an agent in the prevention of organ transplant rejection and graft-versus-host-disease

ABSTRACT

The present invention relates to methods of using lactoferrin (LF) to treat, prevent or reduce the incidence of organ transplant rejection and graft-versus-host-disease. More particularly, the present invention relates to methods of reducing an immune response against miss-matched transplanted organs such as kidney, heart, lung, liver, pancreas and stem cells by administering a composition of lactoferrin to the recipient patients. In addition, this invention relates to the treatment of bone marrow transplant (BMT) donors with lactoferrin to attenuate the development of graft-versus-host-disease in the recipients. Moreover, this invention relates to the treatment of xenograft organ donors with lactoferrin to attenuate the development of graft rejection in the recipients.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/432,113 filed Dec. 10, 2002 and 60/498,338 filed Aug. 27, 2003,which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to methods of using lactoferrin(LF) to treat, prevent or reduce the incidence of organ transplantrejection and graft-versus-host-disease (GVHD). More particularly, thepresent invention relates to methods of reducing an immune responseagainst miss-matched transplanted organs such as kidney, heart, lung,liver, pancreas, bone marrow and peripheral stem cells by administeringa composition of lactoferrin to the recipient patients. Moreover, thisinvention relates to the treatment of bone marrow or peripheral stemcell transplant donors with lactoferrin to attenuate the development ofgraft-versus-host-disease in the recipients.

BACKGROUND OF THE INVENTION

[0003] According to current estimates, 80,764 people in America arewaiting for organ transplantation. All organ-transplanted patientsundergo an extensive immunosuppresion therapy with drugs, such ascyclosporine (Neoral®, Sandimmune®), prednisone (Novo Prednisone®, ApoPrednisone®), azathioprine. (Imuran®), tacrolimus or FK506 (Prograf®),mycophenolate mofetil (CellCept®), OKT3 (Muromorab CO3®, Orthoclone®),or ATGAM & Thymoglobulin. While the use of these drugs has improved thechances for survival in patients receiving organ transplants, three-yearmortality rates range from 10-40% and chronic rejection remains aserious issue.

[0004] Almost all patients experience at least one episode of rejectionfollowing transplant surgery. Chronic rejection is a slow, progressiveprocess that usually begins inside the transplant organ's blood vessels,which are lined by donor cells that interact with host white blood cellsin the bloodstream. Over time, as a result of inflammation and rejectionreactions, scar tissue can accumulate inside these vessels, reducing orpreventing blood flow into the filter and chemical plant portions of thekidney. If blockages become widespread, the organ becomes compromisedowing to lack of oxygen and nutrients. Approximately 10 percent ofkidney transplants fail each year due to chronic rejection, graftdysfunction and kidney toxicity, causing the patient to need dialysisand often a new organ.

[0005] While the current therapies used to combat rejection haveresulted in improved transplant outcomes, they are only effective ifused on a continuous basis. Consequently, most patients must maintaintheir regimen of anti-rejection drugs for the rest of their lives. Inaddition, many of the currently approved drugs are highly toxic andproduce severe adverse side effects when used regularly leading to ahigh rate of patient morbidity following the transplant. Adverse effectsfrom these drugs may include fever, nausea, edema, and a wide range ofspecific organ toxicity. Because anti-rejection drugs areimmunosuppressive, patients also run a high risk of developingconcomitant infections. Adverse drug interactions are also common andadministration of current medications must be carefully monitored andcontrolled. Thus, there is a significant unmet need for improvedanti-rejection drugs which demonstrate both efficacy and betterlong-term tolerability.

[0006] One key strategy for reducing the risk of organ transplantrejection is to more effectively modulate the body's natural immuneresponse to the new organ. Recent therapeutic strategies to combat organallograft rejection have focused on T cell signaling pathways and themolecules that comprise them. While some pharmaceuticals have shownpromise in blocking acute rejection, chronic graft destruction andpermanent allograft acceptance in the absence of continuous immunesuppression is problematic. Recent data now suggest T cells hold the keyto generation of transplantation tolerance and alleviation of long-termdrug intervention.

[0007] Lactoferrin is a single chain metal binding glycoprotein. Manycell types, such as monocytes, macrophages, lymphocytes, andbrush-border cells in the intestine, are known to have lactoferrinreceptors. Lactoferrin is found mainly in external secretions of mucosalepithelia such as breast milk, saliva, tears, bile, and pancreatic fluidand has a wide array of functions related to host immune responsemechanisms. For example, lactoferrin has been reported to modulate anumber of key cytokines, chemokines or other molecules that control theimmune response including IL-2, IL-10, IL-18, INF-γ, TNF-α, MIP3-α andNF Kappa-B among others. In addition, lactoferrin has been shown tomodulate the Th1/Th2 immune response pathways as well as activatepolymorphonuclear neutrophils (PMN) and regulate granulopoeisis.

[0008] Recombinant human lactoferrin has previously been described asbeing purified after expression in a variety of prokaryotic andeukaryotic organisms including aspergillus (U.S. Pat. No. 6,080,559),cattle (U.S. Pat. No. 5,919,913), rice, corn, Sacharomcyes (U.S. Pat.No. 6,228,614) and Pichia pastoris (U.S. Pat. Nos. 6,455,687, 6,277,817,6,066,469). Also described are expression systems for the expression offull-length human lactoferrins (e.g., U.S. Pat. No. 6,100,054). In allcases, part of the teaching is expression of the full length cDNA andpurification of the intact protein whose N-terminal, after processing ofthe leader peptide, is the amino acid glycine. Nuijens et al. (U.S. Pat.No. 6,333,311) separately describe variants of human lactoferrin buttheir focus is limited to deletion or substitution of arginine residuesfound in the N-terminal domain of lactoferrin.

[0009] The present invention is the first to use a lactoferrincomposition as a means of imunosuppressant therapy to prevent organrejection and graft-versus-host-disease.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention is directed to a method for modulating animmune response against organ transplants, inducing permanent allograftor xenograft acceptance and reducing the incidence ofgraft-versus-host-disease involved in bone marrow or peripheral stemcells transplantation. The method of treatment involves administrationof a lactoferrin composition.

[0011] The lactoferrin composition, which is dispersed in apharmaceutically acceptable carrier, comprises lactoferrin or N-terminallactoferrin variant in which at least the N-terminal glycine residue istruncated or substituted. The lactoferrin is mammalian lactoferrin, moreparticularly, the lactoferrin is human or bovine. Yet further, thelactoferrin is recombinant lactoferrin. In specific embodiments, theN-terminal lactoferrin variant comprises at least 1% of the lactoferrincomposition, at least 5% of the lactoferrin composition, at least 10% ofthe lactoferrin composition, at least 25% of the lactoferrincomposition, at least 50% of the lactoferrin composition or any range inbetween.

[0012] The amount of the lactoferrin that is administered is about 1 mgto about 20 g per day, more preferably, the amount is about 0.1 g toabout 5 g per day. More particularly, the composition is a solution,capsule or a tablet having a lactoferrin concentration of about 0.1% toabout 100%.

[0013] In further embodiments, a metal chelator dispersed in apharmaceutically acceptable carrier can also be administered with thelactoferrin composition. Preferred metal chelator include, but are notlimited to ethylenediaminetetraacetic acid (EDTA) or[ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA). Morepreferably, the metal chelator is EDTA. The amount of EDTA that isadministered is about 1 ng to about 1 g per day

[0014] A specific embodiment of the present invention is a method oftreating tissue or organ transplant rejection in a recipient comprisingthe step of administering to the recipient a lactoferrin composition inan effective amount to attenuate the tissue or organ transplantrejection. The tissue is bone marrow or peripheral stem cells. Infurther embodiments the organ includes, but is not limited to kidney,heart, lung, liver or pancreas. The lactoferrin composition reducesallogeneic immune responses in the recipient. Still further, thelactoferrin composition regulates T cell responses by inducingtransplant tolerance in the recipient.

[0015] A further embodiment comprises administering anyimmunosuppressive agent, anti-rejection agent or anti-GVHD therapy incombination with the lactoferrin composition. The immunosupressive agentcan be selected from the group consisting of cyclosporine, prednisone,azathioprine, tacrolimus and mycophenolate mofetil.

[0016] In a further embodiment, the lactoferrin composition modulatesthe mucosal or systemic immune system in a subject by increasing theamount of lactoferrin in the gastrointestinal tract, for examplestimulating interleukin-18 or MIP-3-alpha in the gastrointestinal tract.The lactoferrin composition can also regulate the activity of immunecells, for example, but not limited to B and T lymphocytes (e.g.,CD4+/CD3+ and CD8+/CD3+cells), antigen-presenting cells, natural killercells, macrophages and granulocytes. Still further, the lactoferrincomposition down-regulates the production or activity ofpro-inflammatory cytokines.

[0017] The lactoferrin composition of the present invention can beadministered parenterally, for example, subcutaneously, intramuscularly,intraperitoneally, intravenously, intraarterially, intramyocardially,transendocardially, transepicardially, or intrathecally.

[0018] In a further embodiment, the lactoferrin composition isadministered orally. For oral administration, an antacid in combinationwith the lactoferrin composition can be administered. The lactoferrincan be formulated in a delayed release formulation. Still further, thelactoferrin composition can be formulated wherein release occurs in thesmall intestine or in the large intestine.

[0019] Another embodiment is a method of preventing tissue or organtransplant rejection in a recipient comprising the step of administeringto the recipient a lactoferrin composition in an effective amount toprevent the tissue or organ transplant rejection.

[0020] A further embodiment is a method of modulating the immuneresponse against an organ or tissue transplant in a recipient comprisingthe step of administering to the recipient a lactoferrin composition inan effective mount to modulate the response against the organ or tissuetransplant.

[0021] Still further, another embodiment is a method of treatinggraft-versus-host-disease in a recipient comprising the steps ofadministering to the recipient a lactoferrin composition in an effectiveamount to attenuate the graft-versus-host-disease in the recipient. Theembodiment comprises also treating ex vivo the donor organ or tissuewith the lactoferrin composition. The tissue is a stem cell for example,bone marrow or peripheral stem cells.

[0022] Another embodiment is a method of treatinggraft-versus-host-disease by administering a lactoferrin composition tothe donor organ or donor tissue prior to transplantation into therecipient.

[0023] Another specific embodiment is a method of preventinggraft-versus-host-disease in a recipient comprising the steps ofadministering to the recipient a lactoferrin composition in an amounteffective to prevent the graft-versus-host-disease in the recipient. Theembodiment comprises also treating ex vivo the donor organ or tissuewith the lactoferrin composition. The tissue is a stem cell for example,bone marrow or peripheral stem cells.

[0024] Another embodiment is a method of treating tissue or organtransplant rejection in a recipient comprising the step of administeringto the donor a lactoferrin composition in an amount effective toattenuate the tissue or organ transplant rejection in the recipient.

[0025] Still further, another embodiment is a method of treatingxenograft tissue or xenograft organ transplant rejection in a recipientcomprising the step of administering to the xenograft donor an effectiveamount of a lactoferrin composition to attenuate or prevent the tissueor organ transplant rejection in the recipient.

[0026] The foregoing has outlined rather broadly the features andtechnical advantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated that the conception and specific embodimentdisclosed may be readily utilized as a basis for modifying or designingother structures for carrying out the same purposes of the presentinvention. It should also be realized that such equivalent constructionsdo not depart from the invention as set forth in the appended claims.The novel features which are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] For a more complete understanding of the present invention,reference is now made to the following descriptions taken in conjunctionwith the accompanying drawing.

[0028]FIG. 1 shows that rhLF in combination with FK506 prolongs hearttransplant survival in mice.

[0029]FIG. 2 shows that rhLF prolongs heart allograft survival in mice.

[0030]FIG. 3 shows that rhLF in combination with cyclosporine prolongskidney allograft survival in mice.

DETAILED DESCRIPTION OF THE INVENTION

[0031] It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention disclosed inthis Application without departing from the scope and spirit of theinvention.

[0032] As used herein, the use of the word “a” or “an” when used inconjunction with the term “comprising” in the claims and/or thespecification may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.” Stillfurther, the terms “having”, “including”, “containing” and “comprising”are interchangeable and one of skill in the art is cognizant that theseterms are open ended terms.

[0033] As used herein, the term “allograft” or “allogeneic” refers to agraft that originates from a genetically non-identical donor of the samespecies as the recipient.

[0034] As used herein, the term “autologous” refers to a graft thatoriginates from the recipient.

[0035] The term “chemokine” as used herein refers to small proteins thatare involved in stimulating or inhibiting the migration and activationof cells, for example phagocytic cells and lymphocytes. One of skill inthe art realizes that chemokines play a central role in inflammatory andimmune response processes.

[0036] The term “cytokine” as used herein refers to proteins that aremade by cells that affect the behavior of other cells, for examplestimulate or inhibit cell proliferation. For example, cytokines that aremade by lymphocytes are often called lymphokines or interleukins. One ofskill in the art realizes that the term cytokine is a generic term usedin the literature to refer to proteins that are made by cells that canaffect the behavior of other cells.

[0037] The term “donor” as used herein refers to the subject thatprovides the organ and/or tissue transplant or graft to be transplantedinto the recipient and/or host.

[0038] The term “effective amount” or “therapeutically effective amount”as used herein refers to an amount that results in an improvement orremediation of the symptoms of the disease or condition.

[0039] The term “graft” as used herein refers to the tissue and/or organthat is transplanted or implanted into the host or recipient.

[0040] The term “graft-versus-host-disease” or “GVHD” as used herein isthe pathological reaction that occurs between the host and graftedtissue. The grafted or donor tissue dominates the pathological reaction.Graft-versus-host-disease (GVHD) can be seen following stem cell and/orsolid organ transplantation. GVHD occurs in immunocompromised subjects,who when transplanted, receive “passenger” lymphocytes in thetransplanted stem cells or solid organ. These lymphocytes recognize therecipient's tissue as foreign. Thus, they attack and mount aninflammatory and destructive response in the recipient. GVHD has apredilection for epithelial tissues, especially skin, liver, and mucosaof the gastrointestinal tract. GVHD subjects are immunocompromised duethe fact that prior to transplant of the graft, the subject receivesimmunosuppressive therapy.

[0041] The term “lactoferrin composition” as used herein refers to acomposition having lactoferrin, a portion or part of lactoferrin, anN-terminal lactoferrin variant, or a combination thereof.

[0042] The term “lactoferrin” or “LF” as used herein refers to native orrecombinant lactoferrin. Native lactoferrin can be obtained bypurification from mammalian milk or colostrum or from other naturalsources. Recombinant lactoferrin (rLF) can be made by recombinantexpression or direct production in genetically altered animals, plants,fungi, bacteria, or other prokaryotic or eukaryotic species, or throughchemical synthesis.

[0043] The term “metal chelator” as used herein refers to a compoundwhich binds metal. Metal chelators that can be used in the presentinvention include the divalent metal chelators, for example,ethylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof.

[0044] The term “N-terminal lactoferrin variant” as used herein refersto lactoferrin wherein at least the N-terminal glycine has beentruncated and/or substituted. N-terminal lactoferrin variants alsoinclude, but are not limited to deletion and/or substitution of one ormore N-terminal amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 16 N-terminal amino acid residues, etc.Thus, N-terminal lactoferrin variants comprise at least deletions ortruncations and/or substitutions of 1 to 16 N-terminal amino acidresidues. The deletion and/or substitution of at least the N-terminalglycine of lactoferrin mediates the same biological effects asfull-length lactoferrin and/or may enhance lactoferrin's biologicalactivity, for example by stimulating the production of various cytokines(e.g., IL-18, MIP-3α, GM-CSF or IFN-γ) by inhibiting various cytokines,(e.g., IL-2, IL-4, IL-5, IL-10, or TNF-α), and by improving otherparameters related to allograft organ acceptance and GVHD.

[0045] The term “oral administration” as used herein includes oral,buccal, enteral or intragastric administration.

[0046] The term “organ or tissue transplant rejection” as used hereinrefers to a consequence of organ or tissue transplantation caused by therecipient's or host's immune system in response to the transplantedorgan/tissue, which can damage or destroy it. Thus, one of skill in theart realizes that “organ or tissue transplant rejection” is controlledby the host subject.

[0047] The term “organ” as used herein refers a group of several tissuetypes that perform a given function. Exemplary organs include, but arenot limited to heart, kidney, liver, pancreas, and lung.

[0048] The term “progenitor cell” as used herein refers to a cell thatis an undifferentiated cell that is capable of differentiating. One ofskill in the art realizes that a progenitor cell is an ancestor cell toprogeny descendant cells.

[0049] The term “parenteral administration” as used herein includes anyform of administration in which the compound is absorbed into thesubject without involving absorption via the intestines. Exemplaryparenteral administrations that are used in the present inventioninclude, but are not limited to intramuscular, intravenous,intraperitoneal, intraocular, subcutaneous or intraarticularadministration. Yet further, parenteral administration also includesadministration into a surgical field.

[0050] The term “pharmaceutically acceptable carrier” as used hereinincludes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

[0051] The term “preventing” as used herein refers to minimizing,reducing or suppressing the risk of developing a disease state orparameters relating to the disease state or progression or otherabnormal or deleterious conditions.

[0052] The term “recipient” or “host” as used herein refers to anysubject that receives an organ and/or tissue transplant or graft.

[0053] The term “stem cells” as used herein refers to “undifferentiatedcells” that are capable of proliferation, self-maintenance, productionof differentiated cells or regeneration of stem cells may be tissue. Inpreferred embodiments of the present invention, a stem cell is capableof differentiating into a differentiated cell.

[0054] The term “subject” as used herein, is taken to mean any mammaliansubject to which a human or bovine lactoferrin composition isadministered according to the methods described herein. Thus, a skilledartisan realizes that a mammalian subject, includes, but is not limitedto humans, monkeys, horses, pigs, cows, dogs, cats, rats and mice. In aspecific embodiment, the methods of the present invention are employedto treat a human subject. Another embodiment includes treating a humansubject with organ or bone marrow or peripheral stem cells transplantedaccording to the then medically established guidelines.

[0055] The terms “Th1 cells” and “Th2 cells” as used herein refer tocells defined as subsets of CD4 T-cells that are characterized by thecytokines they produce. These cells are often called helper T-cells. Itis also known that extracellular antigens tend to stimulate theproduction of Th2 cells and intracellular antigens induce Th1 cells.Thus, as used herein, “Th cells” is interchangeable with “helperT-cells”. The terms “Th1 cytokines” and “Th2 cytokines” or “Th1/Th2”refer to the cytokines produced respectively by Th1 or Th2 cells.

[0056] The term “tissue” as used herein refers to a group or collectionof similar cells and the intercellular substance which act together toperform a particular function. Primary tissues include epithelial,connective, muscular and nervous.

[0057] The term “treating” and “treatment” as used herein refers toadministering to a subject a therapeutically effective amount of alactoferrin composition so that the subject has an improvement in theparameters related to allograft organ acceptance and GVHD. Theimprovement is any observable or measurable improvement. Thus, one ofskill in the art realizes that a treatment may improve the patientcondition, but may not be a complete cure of the disease.

[0058] The term “xenogeneic” as used herein refers to grafts, such astissues, cells or organs that are derived from different species.

[0059] The term “xenograft” as used herein refers to a graft thatoriginates from a donor of a species that is different from therecipient. Such donor animal may be genetically modified to reduce theantigenicity of donor organs. Donor species may include pigs andnon-human primates.

[0060] A. Lactoferrin

[0061] The lactoferrin used according to the present invention can beobtained through isolation and purification from natural sources, forexample, but not limited to mammalian milk. The lactoferrin ispreferably mammalian lactoferrin, such as bovine or human lactoferrin.In preferred embodiments, the lactoferrin is produced recombinantlyusing genetic engineering techniques well known and used in the art,such as recombinant expression or direct production in geneticallyaltered animals, plants or eukaryotes, or chemical synthesis. See, forexample, U.S. Pat. Nos. 5,571,896; 5,571,697 and 5,571,691, which areherein incorporated by reference.

[0062] In certain aspects, the present invention provides lactoferrinvariants having enhanced biological activities over natural LF and orrLF, e.g., the ability to stimulate and/or inhibit cytokines orchemokines. In particular, the invention provides variants oflactoferrin from which at least the N-terminal glycine residue has beensubstituted and/or truncated. The N-terminal lactoferrin variants mayoccur naturally or may be modified by the substitution or deletion ofone or more amino acids.

[0063] The deletional variants can be produced by proteolysis oflactoferrin and/or expression of a polynucleotide encoding a truncatedlactoferrin as described in U.S. Pat. No. 6,333,311, which isincorporated herein by reference.

[0064] Substitutional variants or replacement variants typically containthe exchange of one amino acid for another at one or more sites withinthe protein. Substitutions can be conservative, that is, one amino acidis replaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine or methionine;serine to threonine; threonine to serine; tryptophan to tyrosine;tyrosine to tryptophan or phenylalanine; and valine to isoleucine orleucine.

[0065] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

[0066] Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics (Kyte andDoolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0067] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,e.g., still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those that are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

[0068] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

[0069] Still further, it is understood that an amino acid can besubstituted for another having a similar hydrophilicity value and stillobtains a biologically equivalent and immunologically equivalentprotein. In such changes, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those that are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

[0070] Thus, in the present invention, substitutional variants orreplacement can be produced using standard mutagenesis techniques, forexample, site-directed mutagenesis as disclosed in U.S. Pat. Nos.5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377;5,789,166, and 6,333,311, which are incorporated herein by reference. Itis envisioned that at least the N-terminal glycine amino acid residuecan be replaced or substituted with any of the twenty natural occurringamino acids, for example a positively charged amino acid (arginine,lysine, or histidine), a neutral amino acid (alanine, asparagine,cysteine, glutamine, glycine, isoleucine, leucine, methionine,phenylaline, proline, serine, threonine, tryptophan, tyrosine, valine)and/or a negatively charged amino acid (aspartic acid or glutamic acid).Still further, it is contemplated that any amino acid residue within therange of N1 to N16 can be replaced or substituted. It is envisioned thatat least up to 16 of the N-terminal amino acids residues can be replacedor substituted as long as the protein retains it biological and/orfunctional activity, which is stimulating the production of variouscytokines, (e.g., IL-18, MIP-3α, GM-CSF or IFN-γ) by inhibiting variouscytokines, (e.g., IL-2, IL-4, IL-5, IL-10, and TNF-α) and/or byimproving the parameters related to allograft organ acceptance and GVHD.Thus, the N-terminal lactoferrin variants of the present invention areconsidered functional equivalents of lactoferrin.

[0071] In terms of functional equivalents, it is well understood by theskilled artisan that, inherent in the definition of a “biologicallyfunctional equivalent” protein is the concept that there is a limit tothe number of changes that may be made within a defined portion of themolecule while retaining a molecule with an acceptable level ofequivalent biological activity and/or enhancing the biological activityof the lactoferrin molecule. Biologically functional equivalents arethus defined herein as those proteins in which selected amino acids (orcodons) may be substituted. Functional activity is defined as theability of lactoferrin to stimulate or inhibit various cytokines orchemokines and/or by improving the parameters related to allograft organacceptance and GVHD.

[0072] Still further, the N-terminal amino acid residues can besubstituted with a modified and/or unusual amino acids. A table ofexemplary, but not limiting, modified and/or unusual amino acids isprovided herein below. TABLE 1 Modified and/or Unusual Amino Acids Abbr.Amino Acid Aad 2-Aminoadipic acid BAad 3-Aminoadipic acid BAlabeta-alanine, beta-Amino-propionic acid Abu 2-Aminobutyric acid 4Abu4-Aminobutyric acid, piperidinic acid Acp 6-Aminocaproic acid Ahe2-Aminoheptanoic acid Aib 2-Aminoisobutyric acid BAib 3-Aminoisobutyricacid Apm 2-Aminopimelic acid Dbu 2,4-Diaminobutyric acid Des DesmosineDpm 2,2′-Diaminopimelic acid Dpr 2,3-Diaminopropionic acid EtGlyN-Ethylglycine EtAsn N-Ethylasparagine Hyl Hydroxylysine AHylallo-Hydroxylysine 3Hyp 3-Hydroxyproline 4Hyp 4-Hydroxyproline IdeIsodesmosine Aile allo-Isoleucine MeGly N-Methylglycine, sarcosine MeIleN-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methylvaline NvaNorvaline Nle Norleucine Orn Ornithine

[0073] The presence and the relative proportion of an N-terminallactoferrin variants (deletions and/or substitutions) in a preparationof lactoferrin (lactoferrin composition) may be done by determination ofthe N-terminal amino acid sequence by the process of Edman degradationusing standard methods. A relative proportion of N-terminal lactoferrinvariant comprises at least 1% of the lactoferrin composition, at least5% of the lactoferrin composition, at least 10% of the lactoferrincomposition, at least 25% of the lactoferrin composition, at least 50%of the lactoferrin composition or any range in between.

[0074] In this method, the protein is reacted with phenylisothiocyanate(PITC), which reacts with the amino acid residue at the amino terminusunder basic conditions to form a phenylthiocarbamyl derivative(PTC-protein). Trifluoroacetic acid then cleaves off the first aminoacid as its anilinothialinone derivative (ATZ-amino acid) and leaves thenew amino terminus for the next degradation cycle.

[0075] The percentage of N-terminal lactoferrin variant may also be donemore precisely by using a Dansylation reaction. Briefly, protein isdansylated using Dansyl chloride reacted with the protein in alkalineconditions (pH 10). Following the Dansylation, the reaction mixtures aredried to pellets, then completely hydrolyzed in 6N HCl. The proportionof N-terminal amino acids are identified by RP HPLC using an in-linefluorometer in comparison with standards made up of known dansylatedamino acids.

[0076] B. Pharmaceutical Compositions

[0077] The present invention is drawn to a composition comprisinglactoferrin that is dispersed in a pharmaceutical carrier. Thelactoferrin that is contained in the composition of the presentinvention comprises lactoferrin or an N-terminal lactoferrin variant inwhich at least the N-1 terminal glycine residue is truncated orsubstituted. N-terminal lactoferrin variants include variants that atleast lack the N-terminal glycine residue or contain a substitution atthe N-terminal glycine residue. The substitution can comprisesubstituting a natural or artificial amino acid residue for theN-terminal glycine residue. For example, the substitution can comprisesubstituting a positive amino acid residue or a negative amino acidresidue for the N-terminal glycine residue or substituting a neutralamino acid residue other than glycine for the N-terminal glycineresidue. Other N-terminal lactoferrin variants include lactoferrinlacking one or more N-terminal residues or having one or moresubstitutions in the N-terminal. The N-terminal lactoferrin variantcomprises at least 1% of the composition, at least 5% of thecomposition, at least 10% of the composition, at least 25% of thecomposition, at least 50% of the composition or any range in between.

[0078] Yet further, the composition comprises lactoferrin in combinationwith a metal chelator dispersed in a pharmaceutical carrier. Thus, thepresent invention is drawn to a lactoferrin composition with or withouta metal chelator that is dispersed in a pharmaceutical carrier. One ofskill in the art understands that both compositions (e.g., lactoferrinalone or lactoferrin in combination with a metal chelator) are withinthe scope of the present invention and can be used interchangeablydepending upon the type of response that is desired. It is envisionedthat the addition of a metal chelator to the lactoferrin compositionenhances the sequestering of metal ions and thus strengthens the immunesystem or enhances the effect of lactoferrin.

[0079] Metal chelators that can be used in combination with lactoferrin,include the divalent metal chelators, for example,ethylenediaminetetraacetic acid (EDTA),[ethylenebis(oxyethylenenitrilo)] tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or any salts thereof.More preferably, EDTA is used in combination with lactoferrin.

[0080] Further in accordance with the present invention, the compositionof the present invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, e.g.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof.

[0081] In accordance with the present invention, the composition iscombined with the carrier in any convenient and practical manner, e.g.,by solution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

[0082] In a specific embodiment of the present invention, thecomposition is combined or mixed thoroughly with a semi-solid or solidcarrier. The mixing can be carried out in any convenient manner such asgrinding. Stabilizing agents can be also added in the mixing process inorder to protect the composition from loss of therapeutic activity,e.g., denaturation in the stomach. Examples of stabilizers for use in anthe composition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc., proteolytic enzymeinhibitors, and the like. Yet further, it is envisioned that divalentmetal chelators, for example EDTA, can also be used to stabilize thecomposition of the present invention. More preferably, for an orallyadministered composition, the stabilizer can also include antagonists tothe secretion of stomach acids.

[0083] The composition for oral administration which is combined with asemi-solid or solid carrier can be further formulated into hard or softshell gelatin capsules, tablets, or pills. More preferably, gelatincapsules, tablets, or pills are enterically coated. Enteric coatingsprevent denaturation of the composition in the stomach or upper bowelwhere the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Uponreaching the small intestines, the basic pH therein dissolves thecoating and permits the lactoferrin composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells.

[0084] In another embodiment, a powdered composition is combined with aliquid carrier such as, e.g., water or a saline solution, with orwithout a stabilizing agent.

[0085] The compositions of the present invention may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

[0086] Sterile injectable solutions are prepared by incorporating thelactoferrin in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0087] Further, the composition for topical administration which iscombined with a semi-solid carrier can be further formulated into a gelointment. A preferred carrier for the formation of a gel ointment is agel polymer. Preferred polymers that are used to manufacture a gelcomposition of the present invention include, but are not limited tocarbopol, carboxymethyl-cellulose, and pluronic polymers. Gel polymersprevent denaturation of the composition in the open skin by serumproteases.

[0088] The amount of lactoferrin in the present invention may vary fromabout 1 ng to about 100 g of lactoferrin, more preferably 1 mg to 20 gper day, most preferably 0.1 g to 5 g. In preferred embodiments, thecomposition of the present invention comprises a lactoferrinconcentration of about 0.1% to about 100%. The lactoferrin compositionmay comprise lactoferrin or an N-terminal lactoferrin variant in whichat least the N-1 terminal glycine residue is truncated and/orsubstituted.

[0089] More preferably, the composition of the present invention alsocontains metal chelators, for example, but not limited toethylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof. Theamount of the metal chelator in the composition may vary from about 1 ngto about 1 g. A preferred metal chelator is EDTA.

[0090] Upon formulation, solutions are administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective to result in an improvement or remediation ofthe symptoms. The formulations are easily administered in a variety ofdosage forms such as ingestible solutions, drug release capsules and thelike. Some variation in dosage can occur depending on the condition ofthe subject being treated. The person responsible for administrationcan, in any event, determine the appropriate dose for the individualsubject.

[0091] C. Treatment of Organ Transplant Rejection and GVHD

[0092] In accordance with the present invention, the compositionprovided in any of the above-described pharmaceutical carriers isadministered as a therapeutic drug to a subject who has undergone organtransplant (kidney, heart, lung, liver, pancreas, bone marrow,peripheral stem cells) and/or as a prophylactic drug to organ and/ortissue transplant, for example bone marrow (BM) or peripheral stem celldonor cells, to prevent the development of GVHD in recipients and/or toprevent or attenuate transplant rejection in the recipients. One ofskill in the art can determine the patients who would potentiallybenefit from a therapeutic agent that would reduce chronic allograftrejection and toxicity associated with standard therapy, or thedevelopment of GVHD. One of skill in the art can determine thetherapeutically effective amount of the composition to be administeredto a subject based upon several considerations, such as local effects,pharmacodynamics, absorption, metabolism, method of delivery, age,weight, disease severity and response to the therapy.

[0093] A further embodiment is treating, preventing or reducing the riskof developing graft-versus-host-disease by administering a lactoferrincomposition to the donor organ or donor tissue prior to transplantationinto the recipient. It is envisioned that administering the lactoferrincomposition to the donor tissue/organ will attenuate the immune cells inthe donor/organ and prevent the development of the immune response thatis mounted against the recipient's tissue, thus preventing orattenuating GVHD. In further embodiments, the recipient and the donororgan/tissue can be treated with the lactoferrin composition of thepresent invention.

[0094] Additional embodiments of the present invention include a methodof treating, preventing or attenuating the severity of tissue or organtransplant rejection in a recipient comprising the step of administeringto the donor a lactoferrin composition in an amount sufficient toattenuate the tissue or organ transplant rejection in the recipient. Itis envisioned that the lactoferrin composition reduces allogeneic immuneresponses in the recipient. The lactoferrin composition can also beadministered to both the recipient and the donor.

[0095] Another embodiment is a method of treating, preventing orattenuating the severity of xenograft tissue or xenograft organtransplant rejection in a recipient comprising the step of administeringto the xenograft donor a lactoferrin composition in an amount sufficientto attenuate the tissue or organ transplant rejection in the recipient.The lactoferrin composition can also be administered to both therecipient and the xenograft donor.

[0096] In particular embodiments, progenitor cells or stem cells may beisolated from at least one organ, cell, tissue or organism. Stem cellscan be isolated from embryonic or nonembryonic donors. The tissues fromwhich the stem cells can be isolated include, for example, but are notlimited to the bone marrow, the spleen, the liver, peripheral blood,umbilical cord tissue, umbilical cord blood, adipose tissue or skin. Yetfurther, tissue grafts may be used in the present invention. The tissuemay be part or separated from an organism. In certain embodiments, atissue may comprise, but is not limited to, adipocytes, alveolar,ameloblasts, axon, basal cells, blood (e.g., lymphocytes), blood vessel,bone, bone marrow, peripheral stem cells, brain, breast, cartilage,cervix, colon, cornea, embryonic, endometrium, endothelial, epithelial,esophagus, facia, fibroblast, follicular, ganglion cells, glial cells,goblet cells, kidney, liver, lung, lymph node, muscle, neuron, ovaries,pancreas, peripheral blood, prostate, skin, skin, small intestine,spleen, stem cells, stomach, testes, or ascite tissue.

[0097] In specific embodiments, an organ is the graft. Organs arecomprised of tissues having a special function. Exemplary organs thatare used in grafts in the present invention include, but are not limitedto heart, kidney, pancreas, lung, or liver.

[0098] Oral administration of the composition includes oral, buccal,enteral or intragastric administration. It is also envisioned that thecomposition may be used as a food additive. For example, the compositionis sprinkled on food or added to a liquid prior to ingestion. In furtherembodiments, the composition is administered in conjunction with anantacid. Thus, an antacid is administered prior or substantiallysimultaneously with or after oral administration of the composition. Theadministration of an antacid just prior or immediately following theadministration of the composition may help to reduce the degree ofinactivation of the lactoferrin in the digestive tract. Examples ofappropriate antacids include, but are not limited to, sodiumbicarbonate, magnesium oxide, magnesium hydroxide, calcium carbonate,magnesium trisilicate, magnesium carbonate and alumin hydroxide gel.

[0099] In addition to oral administration, the lactoferrin compositioncan also be administered parenterally, which includes, but is notlimited to intradermal, subcutaneous, intramuscular, intraperitoneal,intravenous, intraarterial, intramyocardial, transendocardial,transepicardial, intrathecal, and infusion techniques.

[0100] In a preferred embodiment of the present invention, thecomposition is administered in an effective amount to decrease, reduce,inhibit or abrogate chronic allograft rejection and toxicity related tostandard therapy. The amount of lactoferrin in the composition may varyfrom about 1 ng to about 100 g, more preferably, 1 mg to about 20 g.Preferably, the composition that is orally administered contains therange of 0.1 g to 5 g of lactoferrin per day.

[0101] In further embodiments, the composition of the present inventionalso contains metal chelators, for example, but not limited toethylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof. Theamount of the metal chelator in the composition may vary from about 1 ngto about 1 g. A preferred metal chelator is EDTA.

[0102] Treatment regimens may vary as well, and often depend on thehealth and age of the patient. Obviously, certain types of disease willrequire more aggressive treatment, while at the same time, certainpatients cannot tolerate more taxing regimens. The clinician will bebest suited to make such decisions based on the known efficacy andtoxicity (if any) of the therapeutic formulations.

[0103] In specific embodiments, the composition is given in a singledose or multiple doses. The single dose may be administered daily, ormultiple times a day, or multiple times a week, or monthly or multipletimes a month. In a further embodiment, the lactoferrin is given in aseries of doses. The series of doses may be administered daily, ormultiple times a day, weekly, or multiple times a week, or monthly, ormultiple times a month.

[0104] The improvement is any observable or measurable improvement.Thus, one of skill in the art realizes that a treatment may improve thepatient or subject's condition, but may not be a complete cure of thedisease. In certain aspects, the composition is administered in aneffective amount to decrease, reduce, inhibit or abrogate levels of animmune response against a graft in the recipient. In further aspects, animprovement can consist of any of the following, for example, increasedfunction of the graft, for example, increased urine output for kidneygrafts or decreased jaundice for liver grafts; reduction ininflammation; reduction in general discomfort of the recipient; anoverall increased tolerance for the graft. Thus, after administration oflactoferrin, if any of the above conditions improve, then the amount oflactoferrin is considered to be an effective amount.

[0105] An improvement in GHVD is also any observable or measurableimprovement. Thus, one of skill in the art realizes that a treatment mayimprove the patient or subject's condition, but may not be a completecure of the disease. In certain aspects, the composition is administeredin an effective amount to decrease, reduce, inhibit or abrogate levelsof immune response from the donor's cells, tissue and/or organ againstthe host's tissues. GVHD can be acute or chronic or mild or severe.Improvements in acute symptoms include any of the following, forexample, decrease skin rash, decrease diarrhea, increase in liverfunction, decrease in susceptibility to infection. Improvements inchronic symptoms include, but are not limited to decrease skin rash,decrease dermatitis, decrease hair loss, decrease liver damage, decreasedry eyes and mouth, decrease susceptibility to infections and decreaselung and/or gastrointestinal disorders. Thus, after administration oflactoferrin, if any of the above conditions improve, then the amount oflactoferrin is considered to be an effective amount.

[0106] Still yet, a further embodiment is a method of regulating amucosal immune response in the gastrointestinal tract or a systemicimmune response in a subject comprising the step of administering alactoferrin composition to said subject the composition of the presentinvention. It is envisioned that lactoferrin stimulates MIP-3 α andinterleukin-18 in the gastrointestinal tract, which regulates immuneresponses. For example, interleukin-18 modulates both Th1/Th2 responses.It is known by those of skill in the art that IL-18 plays an importantrole in allogeneic stem cell transplantation. Pre-treatment ofallogeneic donors with IL-18 significantly improves survival and reducesclinical and pathological indices of acute GVHD in BMT recipients. Othercytokines may also be enhanced or repressed for example, but not limitedto IL-1, IL-2, IL-6, IL10, IL-12 and GM-CSF. It is also envisioned thatlactoferrin stimulates Th1/Th2 type-responses through the inductionand/or repression of Th1 and Th2 cytokines.

[0107] D. Combination Treatments

[0108] In order to increase the effectiveness of oral administration ofthe composition of the present invention, it is desirable to combinethese compositions with standard therapy. For example, knownimmunosuppressant agents are used in combination with the composition ofthe present invention. Exemplary agents known to prevent organ rejectionare T cell modifiers such as cyclosporine (Neoral®, Sandimmune®),prednisone (Novo Prednisone®, Apo Prednisone®), azathioprine (Imuran®),tacrolimus or FK506 (Prograf®), mycophenolate mofetil (CellCept®), OKT3(Muromorab CO3®, Orthoclone®), ATGAM® & Thymoglobulin® orserine-threonine phosphatase calcineurin (CN) inhibitors. In specificembodiments, the standard or approved treatment of GVHD, which is highdoses corticosteroids, primarily high-dose methylprednisolone, is usedin combination with the lactoferrin composition of the presentinvention.

[0109] In addition to immunosuppressant other anti-rejection and/oranti-GVHD therapies can be used in combination with the lactoferrincomposition of the present invention. For example, therapies forpreconditioning and prophylaxis of GVHD include, but are not limited tototal body irradiation, cytosine arabinoside, L-phenylalanine mustard,cyclophosphamide, etoposide, triethylene thiophosphoramide,antithymocyte globulin, bisulfan, tacrolimus, methylprednisolone,cyclosporin, or methotrexate. Experimental therapies for treatment ofGVHD include, but are not limited to cytokine inhibitors/antagonists(e.g., anti-TNFαantibody); IL-1 receptor antagonist; recombinant IL-1receptor; inhibitors of T-cell activation (e.g., tacrolimus);antimetabolites (e.g., mycophenolate mofetil); anti-CD3 antibody(muromonab, OKT3); anti-CD25 antibody; anti-IL2 receptor monoclonalantibody daclizumab; extracorporeal photopheresis using ex vivo8-methoxypsoralen; anti-thymocyte globulins (Thymoglobulin® or ATGAM®);ABX-CBL or CBL-1; or Visilizumab (Nuvion®).

[0110] The composition of the present invention may precede, beco-current with and/or follow the other agent(s) by intervals rangingfrom minutes to weeks. In embodiments where the composition of thepresent invention, and other agent(s) are applied separately to a cell,tissue or organism, one would generally ensure that a significant periodof time did not expire between the time of each delivery, such that thecomposition and agent(s) would still be able to exert an advantageouslycombined effect on the cell, tissue or organism.

[0111] Various combination regimens of the composition and one or moreagents are employed. One of skill in the art is aware that thecomposition of the present invention and agents can be administered inany order or combination.

E. EXAMPLES

[0112] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 RhLF in Combination with Immunosuppressive Drugs Delays HeartTransplant Rejection

[0113] Hearts were removed from Buffalo rats and transplanted into theabdomen of WF rats using micro vascular surgery. The survival of thetransplanted heart was measured daily by palpating for its beat.Transplanted animals either remained untreated (n=5) or were treatedwith FK506 (0.8 mg/kg once daily×7 days) plus placebo (n=4) or withFK506 (0.8 mg/kg once daily×7 days) plus rhLF (625 mg/kg b.i.d.×14 daysfollowed by 625 mg/kg once daily till rejection; n=7). Drug treatmentwas started two days prior to the day of transplantation. Averagetransplant survival of animals treated with FK506 alone (20 days) wasnot different from animals receiving FK506+placebo (19.3 days) as shownin FIG. 1. RhLF+FK506 was highly statistically different (p<0.01)relative to FK506 alone or FK506+placebo. (See FIG. 1).

Example 2 Recombinant Human Lactoferrin (rhLF) Monotherapy ProlongsAllograft Survival

[0114] Heterotopic heart transplantation in 8-10 weeks old rats (BUF,donor to WF, recipient) was performed using standard microsurgicaltechnique of end-to-side anastomoses to recipient aorta and vena cava.Graft survival was defined as the last day of transabdominally palpablecardiac contractions. Recipients were treated with either placebo orrhLF (625 mg/Kg) for 14 days starting seven days prior to thetransplant. FIG. 2 illustrates that lactoferrin alone significantlyextended cardiac allograft survival.

Example 3 RhLF Inhibits Kidney Transplant Rejection

[0115] The activity of rhLF in combination with cyclosporine (CsA) wasdetermined on kidney allograft survival model in rats. Untreated ACIrecipients (RT1a) of Lewis (LEW; RT11) kidney allografts survived a meansurvival time (MST) of 8.4±0.6 days (n=5). A 14-day treatment with 1.25mg/kg CsA extended the survival of kidney allografts to 23.0±2.3 days(n=5). FIG. 3 shows that additional treatment of recipients with oralrhLF on days 3 to 14 with 1250 mg/kg and thereafter with 625 mg/kgprolonged survival to 30.6±3.6 days (n=7; p<0.036). These resultsdemonstrate that additional therapy with lactoferrin provided abeneficial effect for kidney allograft survival.

Example 4 Pre-Treatment of Donors with Recombinant Human LactoferrinAttenuates Acute Graft-Versus-Host-Disease

[0116] The effect of pre-treating bone marrow transplantation donorswith rhLF on the severity of acute GVHD is tested in a wellcharacterized experimental Balb/c (H2d) in B6 (H2b) GVHD model. Briefly,2×10⁶ purified donor T cells and 5×10⁶ BM cells from Balb/c mice areinjected into TBI (1300 cGy, split into 2 doses separated by 3 h) B6mice. Donor mice are treated with rhLF (1000 mg/kg) for 8 days before BMtransplantation. Pre-treatment of allogeneic BMT donors with rhLFsignificantly improves survival and reduces clinical and pathologicindices of acute GVHD in BMT recipients.

Example 5 Pre-Treatment of Donor Cell Exvivo with Recombinant HumanLactoferrin Attenuates Acute Graft-Versus-Host-Disease

[0117] The effect of pre-treating bone marrow transplantation donorswith rhLF on the severity of acute GVHD is tested in a wellcharacterized experimental Balb/c (H2d) in B6 (H2b) GVHD model. Briefly,2×10⁶ purified donor T cells and 5×10⁶ BM cells from Balb/c mice arepreincubated with rhLF at 10 mg/ml for 3 hours, then infused into B6mice receiving TBI (1300 cGy, split into 2 doses separated by 3 h).Pre-treatment of allogeneic BMT donor cells with rhLF significantlyimproves survival and reduces clinical and pathologic indices of acuteGVHD in BMT recipients.

Example 6 Allograft Survival Following Organ Treatment is Extended byEx-Vivo Treatment with rhLF

[0118] Heterotopic heart transplantation in 8-10 weeks old rats (WF,donor to BUF, recipient) is performed using standard microsurgicaltechnique of end-to-side anastomoses to recipient aorta and vena cava.Graft survival is defined as the last day of transabdominally palpablecardiac contractions. Donor hearts are isolated and pretreated exvivowith placebo or rhLF at 1 mg/ml or 10 mg/ml for 2 hours or 6 hoursbefore transplantation. The data show that pretreatment of thetransplanted organ with lactoferrin significantly extends cardiacallograft survival.

Example 7 Pre-Treatment of Donors with Recombinant Human LactoferrinExtends Heart Allograft Survival

[0119] Heterotopic heart transplantation in 8-10 weeks old rats (WF,donor to BUF, recipient) is performed using standard microsurgicaltechnique of end-to-side anastomoses to recipient aorta and vena cava.Graft survival is defined as the last day of transabdominally palpablecardiac contractions. Donor mice are treated with rhLF (1000 mg/kg) for8 days before organ transplantation. Pre-treatment of allogeneic organdonors with rhLF significantly improves allograft survival.

Example 8 RhLF Improves Kidney Allograft Acceptance

[0120] Human patients with kidney transplants are administered rhLF orplacebo. The incidence of biopsy-confirmed acute rejection is testedduring the first 6 and 12 months post-transplantation and patientsurvival and graft survival is measured at 12 monthspost-transplantation.

Example 9 RhLF Improves Kidney Allograft Acceptance in CombinationTherapy

[0121] Human patients with kidney transplants are administered eithercyclosporine plus either rhLF or placebo. The incidence ofbiopsy-confirmed acute rejection is tested during the first 6 and 12months post-transplantation and patient survival and graft survival ismeasured at 12 months post-transplantation. The incidence and severityof renal failure is also measured and compared between the rhLF andplacebo treated groups.

Example 10 RhLF Improves Kidney Allograft Acceptance in CombinationTherapy

[0122] Human patients with kidney transplants are administered eitherthe standard clinical dose of cyclosporine or dose-reduced cyclosporineplus rhLF. The incidence of biopsy-confirmed acute rejection is testedduring the first 6 and 12 months post-transplantation and patientsurvival and graft survival is measured at 12 monthspost-transplantation. The incidence and severity of renal failure isalso measured and compared between the two groups.

Example 11 RhLF Improves Heart Allograft Acceptance

[0123] Human patients with heart transplants are administered rhLF orplacebo. The incidence of biopsy-confirmed acute rejection is testedduring the first 6 and 12 months post-transplantation and patientsurvival and graft survival is measured at 12 monthspost-transplantation.

Example 12 RhLF Improves Liver Allograft Acceptance

[0124] Human patients with liver transplants are administered rhLF orplacebo. The incidence of biopsy-confirmed acute rejection is testedduring the first 6 and 12 months post-transplantation and patientsurvival and graft survival is measured at 12 monthspost-transplantation.

Example 13 RhLF Improves Lung Allograft Acceptance

[0125] Human patients with lung transplants are administered rhLF orplacebo. The incidence of biopsy-confirmed acute rejection is testedduring the first 6 and 12 months post-transplantation and patientsurvival and graft survival is measured at 12 months.

Example 14 RhLF Improves Pancreas Allograft Acceptance

[0126] Human patients with pancreas transplants are administered rhLF orplacebo. The incidence of biopsy-confirmed acute rejection is testedduring the first 6 and 12 months post-transplantation and patientsurvival and graft survival is measured at 12 months.

Example 15 RhLF Treatment of Donors Improves Kidney Allograft Acceptance

[0127] Human kidney donors are treated with rhLF 3 mg bid for 14 daysbefore harvesting of kidney for transplant. The incidence ofbiopsy-confirmed acute rejection is tested in the recipients during thefirst 6 and 12 months post-transplantation and patient survival, graftsurvival is measured at 12 months post-transplantation and incidence anddose of cylcosporin use is measured at 12 months.

Example 16 Treatment of Recipients with Recombinant Human LactoferrinAttenuates Acute Graft-Versus-Host-Disease

[0128] The effect of prophylactically treating stem cell transplantationrecipients with rhLF on the incidence and severity of acute GVHD istested in a clinical trial. Stem cell recipients receive placebo or rhLF3 mg bid for 30 days starting one day prior to allogeneic stem celltransplantation. Incidence and severity of acute GVHD and use ofsteroids and days of hospitalization are evaluated.

Example 17 Treatment of Recipients with Recombinant Human Lactoferrin inCombination Therapy Attenuates Acute Graft-Versus-Host-Disease

[0129] Patients with acute graft-versus-host-disease are treated withoral corticosteroids plus either rhLF or placebo. The response rate isevaluated in terms of reduction in the severity of acute GVHD, days ofhospitalization, incidence of chronic GVHD and 180-day survival.

Example 18 Pretreatment of Donors with Recombinant Human LactoferrinAttenuates Acute Graft-Versus-Host-Disease

[0130] The effect of pre-treating treating stem cell transplantationdonors with rhLF on the subsequent incidence and severity of acute GVHDin the recipients is tested in a clinical trial. Donors receive placeboor rhLF 3 mg bid for 30 days prior to the harvesting of stem cells.Incidence and severity of acute GVHD and use steroids and days ofhospitalization in the recipients is evaluated.

REFERENCES CITED

[0131] All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

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[0144] Although the present invention and its advantages have beendescribed in detail, it should be understood that various changes,substitutions and alterations can be made herein without departing fromthe invention as defined by the appended claims. Moreover, the scope ofthe present application is not intended to be limited to the particularembodiments of the process, machine, manufacture, composition of matter,means, methods and steps described in the specification. As one willreadily appreciate from the disclosure, processes, machines,manufacture, compositions of matter, means, methods, or steps, presentlyexisting or later to be developed that perform substantially the samefunction or achieve substantially the same result as the correspondingembodiments described herein may be utilized. Accordingly, the appendedclaims are intended to include within their scope such processes,machines, manufacture, compositions of matter, means, methods, or steps.

What is claimed is:
 1. A method of treating tissue or organ transplantrejection in a recipient comprising the step of administering to therecipient an effective amount of a lactoferrin composition to attenuatethe tissue or organ transplant rejection.
 2. The method of sentence 1wherein said lactoferrin composition reduces allogeneic immune responsesin the recipient.
 3. The method of sentence 1, wherein the tissue isbone marrow or peripheral stem cells.
 4. The method of sentence 1,wherein the organ is kidney, heart, lung, liver, or pancreas.
 5. Themethod of sentence 1 wherein said lactoferrin composition regulates Tcell responses by inducing transplant tolerance in the recipient.
 6. Themethod of sentence 1, wherein said lactoferrin composition is dispersedin a pharmaceutically acceptable carrier.
 7. The method of sentence 1,wherein said lactoferrin is mammalian lactoferrin.
 8. The method ofsentence 7, wherein said lactoferrin is human or bovine.
 9. The methodof sentence 1, wherein said lactoferrin is recombinant lactoferrin. 10.The method of claim 9, wherein said lactoferrin composition comprises anN-terminal lactoferrin variant.
 11. The method of claim 10, wherein theN-terminal lactoferrin variant lacks at least the N-terminal glycineresidue.
 12. The method of claim 11, wherein said N-terminal lactoferrinvariant comprises at least 1% to at least 50% of the lactoferrincomposition.
 13. The method of sentence 1, wherein said lactoferrincomposition is administered orally.
 14. The method of sentence 1,wherein said lactoferrin composition is administered parenterally. 15.The method of sentence 13 further comprising administering an antacid inconjunction with said lactoferrin composition.
 16. The method ofsentence 13 further comprising administering the lactoferrin in adelayed release formulation.
 17. The method of sentence 16, wherein thelactoferrin release occurs in the small intestine.
 18. The method ofsentence 16, wherein the lactoferrin release occurs in the largeintestine.
 19. The method of sentence 1, wherein the amount of thelactoferrin composition that is administered is about 1 mg to about 20 gper day.
 20. The method of sentence 1, wherein the amount of thelactoferrin composition that is administered is about 0.1 g to about 5 gper day.
 21. The method of sentence 1, wherein said lactoferrinmodulates the mucosal or systemic immune system in a subject byincreasing the amount of lactoferrin in the gastrointestinal tract. 22.The method of sentence 21, wherein said lactoferrin stimulatesinterleukin-18 or MIP-3-alpha in the gastrointestinal tract.
 23. Themethod of sentence 21, wherein said lactoferrin regulates the activityof immune cells.
 24. The method of sentence 21, wherein the immune cellsare B and T lymphocytes, antigen-presenting cells, natural killer cells,macrophages and granulocytes.
 25. The method of sentence 24, wherein theT lymphocytes are selected from the group consisting of CD4+/CD3+,CD8+/CD3+cells and NK-T cells.
 26. The method of sentence 21, whereinsaid lactoferrin regulates the production or activity ofpro-inflammatory cytokines.
 27. The method of sentence 1 furthercomprising administering a metal chelator dispersed in apharmaceutically acceptable carrier.
 28. The method of sentence 27,wherein the metal chelator is ethylenediaminoetetracacetic acid (EDTA)or [etheylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).
 29. Themethod of sentence 28, wherein the metal chelator is EDTA.
 30. Themethod of sentence 29, wherein the amount of EDTA that is administeredis about 1 ng to about 1 g per day.
 31. The method of sentence 1,further comprising administering an immunosuppressive agent,anti-rejection agent or anti-GVHD therapy in combination with thelactoferrin composition.
 32. The method of sentence 31, wherein theimmunosupressive agent is selected from the group consisting ofcyclosporine, prednisone, azathioprine, tacrolimus and mycophenolatemofetil.
 33. A method of preventing tissue or organ transplant rejectionin a recipient comprising the step of administering to the recipient aneffective amount of a lactoferrin composition to prevent the tissue ororgan transplant rejection.
 34. A method of modulating the immuneresponse against an organ or tissue transplant in a recipient comprisingthe step of administering to the recipient an effective amount of alactoferrin composition to modulate the response against the organ ortissue transplant.
 35. A method of treating graft-versus-host-disease ina recipient comprising the step of administering to the recipient aneffective amount of a lactoferrin composition to attenuate thegraft-versus-host-disease in the recipient.
 36. The method of sentence35 further comprising treating ex vivo the donor organ or tissue withthe lactoferrin composition.
 37. The method of sentence 36, wherein thetissue is a stem cell.
 38. The method of sentence 36, wherein the tissueis bone marrow or peripheral stem cells.
 39. A method of treatinggraft-versus-host-disease comprising the step of administering aneffective amount of a lactoferrin composition to the donor organ ordonor tissue prior to transplantation into the recipient.
 40. A methodof preventing graft-versus-host-disease in a recipient comprising thestep of administering to the recipient an effective amount of alactoferrin composition to prevent the graft-versus-host-disease in therecipient.
 41. The method of sentence 40 further comprising treating exvivo the donor organ or tissue with the lactoferrin composition.
 42. Themethod of sentence 41, wherein the tissue is a stem cell.
 43. A methodof treating tissue or organ transplant rejection in a recipientcomprising the step of administering to the donor an effective amount ofa lactoferrin composition to attenuate the tissue or organ transplantrejection in the recipient.
 44. A method of treating xenograft tissue orxenograft organ transplant rejection in a recipient comprising the stepof administering to the xenograft donor an effective amount of alactoferrin composition to attenuate the tissue or organ transplantrejection in the recipient.